Mitochondrial tRNA can be used in translation by mitochondrial EF-Tu (EF-Tumt), but not by E. coli EF-Tu. We hypothesize that the conformation of mitochondrial tRNA uniquely inhibits its use by E. coli EF-Tu, but that EF-Tumt alters the shape of the tRNA and is able to present it to the ribosome in the proper conformation. Chimeric tRNAPhe will be prepared in which different regions (acceptor stem/loop, D stem/loop, anticodon stem/loop and TpsiC stem/loop) of mitochondrial tRNAPhe will be replaced with E. coli tRNAPhe. Structural characteristics of the chimeric tRNAs will be compared to mitochondrial and E. coli tRNAPhe. The ability of each chimeric tRNA to function at each stage in translation will be assessed using both EF-Tumt and E. coli EF-Tu, to determine which region(s) of the mitochondrial tRNA inhibits use by E. coli EF-Tu. Physical methods including scanning force microscopy and fluoresence labeling will be used to identify changes in the conformation of tRNA when bound to EF-Tu. Point mutations in human tRNALeu lead to a variety of human genetic diseases. Very little is known about the biochemical processes affected by these mutated tRNAs. Transcripts of human tRNALeu containing these mutations will be prepared and structurally analyzed. The ability of the mutants to function at each step in mitochondrial translation will be analyzed to determine how the mutation may lead to a human genetic disease.